Food Safety Testing: Rapid Molecular Methods for Chemical and Biological Hazards

The central goal of food safety policy in the European Union (EU) is to protect consumer health by guaranteeing a high level of food safety throughout the food chain. This goal can in part be achieved by testing foodstuffs for the presence of various chemical and biological hazards. The aim of this study was to facilitate food safety testing by providing rapid and user-friendly methods for the detection of particular food-related hazards. Heterogeneous competitive time-resolved fluoroimmunoassays were developed for the detection of selected veterinary residues, that is coccidiostat residues, in eggs and chicken liver. After a simplified sample preparation procedure, the immunoassays were performed either in manual format with dissociation-enhanced measurement or in automated format with pre-dried assay reagents and surface measurement. Although the assays were primarily designed for screening purposes providing only qualitative results, they could also be used in a quantitative mode. All the developed assays had good performance characteristics enabling reliable screening of samples at concentration levels required by the authorities. A novel polymerase chain reaction (PCR)-based assay system was developed for the detection of Salmonella spp. in food. The sample preparation included a short non-selective pre-enrichment step, after which the target cells were collected with immunomagnetic beads and applied to PCR reaction vessels containing all the reagents required for the assay in dry form. The homogeneous PCR assay was performed with a novel instrument platform, GenomEra^™, and the qualitative assay results were automatically interpreted based on end-point time-resolved fluorescence measurements and cut-off values. The assay was validated using various food matrices spiked with sub-lethally injured Salmonella cells at levels of 1-10 colony forming units (CFU)/25 g of food. The main advantage of the system was the exceptionally short time to result; the entire process starting from the pre-enrichment and ending with the PCR result could be completed in eight hours. In conclusion, molecular methods using state-of-the-art assay techniques were developed for food safety testing. The combination of time-resolved fluorescence detection and ready-to-use reagents enabled sensitive assays easily amenable to automation. Consequently, together with the simplified sample preparation, these methods could prove to be applicable in routine testing.

Effect of physico-chemical conditions and operating parameters on flux and retention of different components in ultrafiltration and nanofiltration fractionation of sweet whey

Tässä väitöstutkimuksessa tutkittiin fysikaaliskemiallisten olosuhteiden ja toimintaparametrien vaikutusta juustoheran fraktiointiin. Kirjallisuusosassa on käsitelty heran ympäristövaikutusta, heran hyödyntämistä ja heran käsittelyä kalvotekniikalla. Kokeellinen osa on jaettu kahteen osaan, joista ensimmäinen käsittelee ultrasuodatusta ja toinen nanosuodatusta juustoheran fraktioinnissa. Ultrasuodatuskalvon valinta tehtiin perustuen kalvon cut-off lukuun, joka oli määritetty polyetyleeniglykoliliuoksilla olosuhteissa, joissa konsentraatiopolariosaatioei häiritse mittausta. Kriittisen vuon konseptia käytettiin sopivan proteiinikonsentraation löytämiseksi ultrasuodatuskokeisiin, koska heraproteiinit ovat tunnetusti kalvoa likaavia aineita. Ultrasuodatuskokeissa tutkittiin heran eri komponenttien suodattumista kalvon läpi ja siihen vaikuttavia ominaisuuksia. Herapermeaattien peptidifraktiot analysoitiin kokoekskluusiokromatografialla ja MALDI-TOF massaspektrometrillä. Kokeissa käytettävien nanosuodatuskalvojen keskimääräinen huokoskoko analysoitiin neutraaleilla liukoisilla aineilla ja zeta-potentiaalit virtauspotentiaalimittauksilla. Aminohappoja käytettiin malliaineina tutkittaessa huokoskoon ja varauksen merkitystä erotuksessa. Aminohappojen retentioon vaikuttivat pH ja liuoksen ionivahvuus sekä molekyylien väliset vuorovaikutukset. Heran ultrasuodatuksessa tuotettu permeaatti, joka sisälsi pieniä peptidejä, laktoosia ja suoloja, nanosuodatettiin happamassa ja emäksisessä pH:ssa. Emäksisissä oloissa tehdyssä nanosuodatuksessa foulaantumista tapahtui vähemmän ja permeaattivuo oli parempi. Emäksisissä oloissa myös selektiivisyys laktoosin erotuksessa peptideistä oli parempi verrattuna selektiivisyyteen happamissa oloissa.

The functional and biological properties of whey proteins : prospects for the development of functional foods

Selostus: Heraproteiinit terveysvaikutteisten elintarvikkeiden kehittämisessä

Rasvaisten ja öljyisten jätevesien biologisen puhdistuksen mallinnus

Työn kirjallisuusosassa on tarkasteltu rasvaistenjätevesien puhdistuksessa käytettyjä perinteisiä käsittelymenetelmiä ja ultrasuodatusta. Perinteisiä rasvaisten jätevesien käsittelymenetelmiä ovat muun muassalaskeutus, flotaatio, hydrosykloni, pisarakoon kasvattaminen suodatus sekä biologinen käsittely. Lisäksi happohydrolyysia voidaan soveltaa edellä mainittujen menetelmien esikäsittelynä. Perinteisten puhdistusmenetelmien käyttöä rajoittavatniiden tehottomuus emulgoituneen ja liukoisen öljyn poistossa. Tämä sekä kiristyneet päästövaatimukset ja kalvotekniikan nopea kehittyminen ovat lisänneet kiinnostusta kalvotekniikkaan. Työn soveltavassa osassa on tarkasteltu rasvojen mahdollisesti aiheuttamia ongelmia Porvoon jalostamon kemiallisessa ja biologisessa puhdistuksessa. Rasvaisia jätevesiä muodostuu biodieselin valmistuksessa, jossa rasvoja käytetään syöttöaineena. Vertailtaessa jalostamon vesilaitoksen nykyisiä olosuhteita ja rasvojen käsittelyn vaatimia olosuhteita havaitaan, että optimiolosuhteet ovat melko lähellä toisiaan ja rasvaisten jätevesien mukana tulevat fosfori-, typpi- ja COD-kuormat melko pieniä. Suurimmat mahdolliset rasvojen aiheuttamat ongelmat syntyvät aktiivilietelaitoksella, jossa kevyt pinnalle nouseva rasva nostaa mukanaan lietettä. Rasvat ja rasvahapot myös lisäävät rihmamaisten bakteerien kasvua, joiden runsas esiintyminen aiheuttaa huonosti laskeutuvaa lietettä, eli paisuntalietettä. Rasvaisten vesien aiheuttamaa kuormitusta aktiivilieteprosessiin on tarkasteltu Activated sludge Model No. 3:n ja bio-P fosforin poisto moduuliin pohjautuvan Excel-taulukkolaskentamallin avulla. Pohjana työssä on käytetty Tuomo Hillin vuonna 2002 diplomityönä tekemää taulukkolaskentamallia. Työssä on esitelty kaikki mallin kannalta oleelliset yhtälöt ja parametrit. Tämän tutkimuksen perusteella mallin käytettävyyttä rajoittaa se, että sitä ei ole kalibroitu Porvoon jalostamolle. Kalibroimattomalla mallilla voidaan saada vain suuntaa antavia tuloksia.

Osteoclastic tartrate-resistant acid phosphatase 5b. Diagnostic use and biological significance in bone physiology

The skeleton undergoes continuous turnover throughout life. In women, an increase in bone turnover is pronounced during childhood and puberty and after menopause. Bone turnover can be monitored by measuring biochemical markers of bone resorption and bone formation. Tartrate-resistant acid phosphatase (TRACP) is an enzyme secreted by osteoclasts, macrophages and dendritic cells. The secreted enzyme can be detected from the blood circulation by recently developed immunoassays. In blood circulation, the enzyme exists as two isoforms, TRACP 5a with an intact polypeptide chain and TRACP 5b in which the polypeptide chain consists of two subunits. The 5b form is predominantly secreted by osteoclasts and is thus associated with bone turnover. The secretion of TRACP 5b is not directly related to bone resorption; instead, the levels are shown to be proportional to the number of osteoclasts. Therefore, the combination of TRACP 5b and a marker reflecting bone degradation, such as C-terminal cross-linked telopeptides of type I collagen (CTX), enables a more profound analysis of the changes in bone turnover. In this study, recombinant TRACP 5a-like protein was proteolytically processed into TRACP 5b-like two subunit form. The 5b-like form was more active both as an acid phosphatase and in producing reactive oxygen species, suggesting a possible function for TRACP 5b in osteoclastic bone resorption. Even though both TRACP 5a and 5b were detected in osteoclasts, serum TRACP 5a levels demonstrated no change in response to alendronate treatment of postmenopausal women. However, TRACP 5b levels decreased substantially, demonstrating that alendronate decreases the number of osteoclasts. This was confirmed in human osteoclast cultures, showing that alendronate decreased the number of osteoclats by inducing osteoclast apoptosis, and TRACP 5b was not secreted as an active enzyme from the apoptotic osteoclasts. In peripubertal girls, the highest levels of TRACP 5b and other bone turnover markers were observed at the time of menarche, whereas at the same time the ratio of CTX to TRACP 5b was lowest, indicating the presence of a high number of osteoclasts with decreased resorptive activity. These results support the earlier findings that TRACP 5b is the predominant form of TRACP secreted by osteoclasts. The major source of circulating TRACP 5a remains to be established, but is most likely other cells of the macrophage-monocyte system. The results also suggest that bone turnover can be differentially affected by both osteoclast number and their resorptive activity, and provide further support for the possible clinical use of TRACP 5b as a marker of osteoclast number.

Surface chemistry of fibres and vessel elements in chemical and recycled pulps

Den viktigaste råvaran i papperstillverkning är pappersmassa. Massan innehåller (ved)fibrer men också finmaterial och andra typers (ved)celler, så som kärlceller. Hur dessa komponenter beter sig under arkformationen i pappersmaskinen eller hur de bidrar till egenskaperna hos det färdiga pappret avgörs till stor del av massakomponenternas ytkemiska sammansättning, fysiska struktur och mängden joniserbara grupper på ytan. I denna avhandling studerades ytegenskaperna hos fraktionerade kemiska massor och returfibermassor med avancerade analystekniker. Rester av avfärgningskemikalier identifierades på både returfibrer och på kärlceller. Dessa kan påverka arkformationen och arkstyrkan på returfiberpapper. Kärlcellernas cellväggsstruktur visade sig skilja sig från fibrernas. Resultaten kan främja utvecklingen av returfiberprosessen och användningen av kärlcellsrika lövvedsmassor.

Fractionation of Hen Egg and Oat Lipids with supercritical Fluids Chemical and functional Properties of Fractions

Hen eggs and oats (Avena Sativa) are important materials for the food industry. Today, instead of merely satisfying the feeling of hunger, consumers are asking for healthier, biologically active and environmentally friendly products. The growing awareness of consumers’ increasing demands presents a great challenge to the food industry to develop more sustainable products and utilise modern and effective techniques. The modification of yolk fatty acid composition by means of feed supplements is well understood. Egg yolk phospholipids are polar lipids and are used in several applications including food, cosmetics, pharmaceuticals, and special nutrients. Egg yolk phospholipids are excellent emulsifiers, typically sold as mixtures of phospholipids, triacylglycerols, and cholesterol. However, highly purified and characterised phospholipids are needed in several sophisticated applications. Industrial fractionation of phospholipids is usually based on organic solvents. With these fractionation techniques, some harmful residues of organic solvents may cause problems in further processing. The objective of the present study was to investigate the methods to improve the functional properties of eggs, to develop techniques to isolate the fractions responsible for the specific functional properties of egg yolk lipids, and to apply the developed techniques to plant-based materials, too. Fractionation techniques based on supercritical fluids were utilised for the separation of the lipid fractions of eggs and oats. The chemical and functional characterisation of the fractions were performed, and the produced oat polar lipid fractions were tested as protective barrier in encapsulation processes. Modifying the fatty acid compositions of egg yolks with different types of oil supplements in feed had no affect on their functional or sensory properties. Based on the results of functional and sensory analysis, it is evident that eggs with modified fatty acid compositions are usable in several industrial applications. These applications include liquid egg yolk products used in mayonnaise and salad dressings. Egg yolk powders were utilised in different kinds of fractionation processes. The precipitation method developed in this study resembles the supercritical anti-solvent method, which is typically used in the pharmaceutical industry. With pilot scale supercritical fluid processes, non-polar lipids and polar lipids were successfully separated from commercially produced egg yolk powder and oat flakes. The egg and oat-based polar lipid fractions showed high purities, and the corresponding delipidated fractions produced using supercritical techniques offer interesting starting materials for the further production of bioactive compounds. The oat polar lipid fraction contained especially digalactosyadiacylglycerol, which was shown to have valuable functional properties in the encapsulation of probiotics.

Long-term nutrient load management and lake restoration: Case of Säkylän Pyhäjärvi (SW Finland)

Eutrophication caused by anthropogenic nutrient pollution has become one of the most severe threats to water bodies. Nutrients enter water bodies from atmospheric precipitation, industrial and domestic wastewaters and surface runoff from agricultural and forest areas. As point pollution has been significantly reduced in developed countries in recent decades, agricultural non-point sources have been increasingly identified as the largest source of nutrient loading in water bodies. In this study, Lake Säkylän Pyhäjärvi and its catchment are studied as an example of a long-term, voluntary-based, co-operative model of lake and catchment management. Lake Pyhäjärvi is located in the centre of an intensive agricultural area in southwestern Finland. More than 20 professional fishermen operate in the lake area, and the lake is used as a drinking water source and for various recreational activities. Lake Pyhäjärvi is a good example of a large and shallow lake that suffers from eutrophication and is subject to measures to improve this undesired state under changing conditions. Climate change is one of the most important challenges faced by Lake Pyhäjärvi and other water bodies. The results show that climatic variation affects the amounts of runoff and nutrient loading and their timing during the year. The findings from the study area concerning warm winters and their influences on nutrient loading are in accordance with the IPCC scenarios of future climate change. In addition to nutrient reduction measures, the restoration of food chains (biomanipulation) is a key method in water quality management. The food-web structure in Lake Pyhäjärvi has, however, become disturbed due to mild winters, short ice cover and low fish catch. Ice cover that enables winter seining is extremely important to the water quality and ecosystem of Lake Pyhäjärvi, as the vendace stock is one of the key factors affecting the food web and the state of the lake. New methods for the reduction of nutrient loading and the treatment of runoff waters from agriculture, such as sand filters, were tested in field conditions. The results confirm that the filter technique is an applicable method for nutrient reduction, but further development is needed. The ability of sand filters to absorb nutrients can be improved with nutrient binding compounds, such as lime. Long-term hydrological, chemical and biological research and monitoring data on Lake Pyhäjärvi and its catchment provide a basis for water protection measures and improve our understanding of the complicated physical, chemical and biological interactions between the terrestrial and aquatic realms. In addition to measurements carried out in field conditions, Lake Pyhäjärvi and its catchment were studied using various modelling methods. In the calibration and validation of models, long-term and wide-ranging time series data proved to be valuable. Collaboration between researchers, modellers and local water managers further improves the reliability and usefulness of models. Lake Pyhäjärvi and its catchment can also be regarded as a good research laboratory from the point of view of the Baltic Sea. The main problem in both of them is eutrophication caused by excess nutrients, and nutrient loading has to be reduced – especially from agriculture. Mitigation measures are also similar in both cases.

Graphene from Graphite by Chemical and Physical Techniques.

Graphene is a material with extraordinary properties. Its mechanical and electrical properties are unparalleled but the difficulties in its production are hindering its breakthrough in on applications. Graphene is a two-dimensional material made entirely of carbon atoms and it is only a single atom thick. In this work, properties of graphene and graphene based materials are described, together with their common preparation techniques and related challenges. This Thesis concentrates on the topdown techniques, in which natural graphite is used as a precursor for the graphene production. Graphite consists of graphene sheets, which are stacked together tightly. In the top-down techniques various physical or chemical routes are used to overcome the forces keeping the graphene sheets together, and many of them are described in the Thesis. The most common chemical method is the oxidisation of graphite with strong oxidants, which creates a water-soluble graphene oxide. The properties of graphene oxide differ significantly from pristine graphene and, therefore, graphene oxide is often reduced to form materials collectively known as reduced graphene oxide. In the experimental part, the main focus is on the chemical and electrochemical reduction of graphene oxide. A novel chemical route using vanadium is introduced and compared to other common chemical graphene oxide reduction methods. A strong emphasis is placed on electrochemical reduction of graphene oxide in various solvents. Raman and infrared spectroscopy are both used in in situ spectroelectrochemistry to closely monitor the spectral changes during the reduction process. These in situ techniques allow the precise control over the reduction process and even small changes in the material can be detected. Graphene and few layer graphene were also prepared using a physical force to separate these materials from graphite. Special adsorbate molecules in aqueous solutions, together with sonic treatment, produce stable dispersions of graphene and few layer graphene sheets in water. This mechanical exfoliation method damages the graphene sheets considerable less than the chemical methods, although it suffers from a lower yield.

New [18F]Tracers for Alzheimer's Disease Imaging. Labeling Synthesis And Biological Testing

Alzheimer’s disease (AD) is the most common form of dementia. Characteristic changes in an AD brain are the formation of β-amyloid protein (Aβ) plaques and neurofibrillary tangles, though other alterations in the brain have also been connected to AD. No cure is available for AD and it is one of the leading causes of death among the elderly in developed countries. Liposomes are biocompatible and biodegradable spherical phospholipid bilayer vesicles that can enclose various compounds. Several functional groups can be attached on the surface of liposomes in order to achieve long-circulating target-specific liposomes. Liposomes can be utilized as drug carriers and vehicles for imaging agents. Positron emission tomography (PET) is a non-invasive imaging method to study biological processes in living organisms. In this study using nucleophilic 18F-labeling synthesis, various synthesis approaches and leaving groups for novel PET imaging tracers have been developed to target AD pathology in the brain. The tracers were the thioflavin derivative [18F]flutemetamol, curcumin derivative [18F]treg-curcumin, and functionalized [18F]nanoliposomes, which all target Aβ in the AD brain. These tracers were evaluated using transgenic AD mouse models. In addition, 18F-labeling synthesis was developed for a tracer targeting the S1P3 receptor. The chosen 18F-fluorination strategy had an effect on the radiochemical yield and specific activity of the tracers. [18F]Treg-curcumin and functionalized [18F]nanoliposomes had low uptake in AD mouse brain, whereas [18F]flutemetamol exhibited the appropriate properties for preclinical Aβ-imaging. All of these tracers can be utilized in studies of the pathology and treatment of AD and related diseases.